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(CANCER RESEARCH 52, 1553-1560, March 15, 1992] Tumor Types Derived from Epithelial and Myoepithelial Rat Mammary Carcinoma1 Cell Lines of R3230AC Anna Sapino, Mauro Papotti, Brunella Sanfìlippo,Patrizia Gugliotta, and Gianni Bussola!i Department of Biomedicai Sciences and Human Oncology, University of Turin, Via Santena 7, Turin, 1-10126, Italy ABSTRACT MATERIALS AND METHODS Epithelial and myoepithelial cells coexist in the rat R3230AC mam mary tumor. To test the hypothesis that these two cell types constitute interactive but independent neoplastic populations, we obtained in vitro cell lines with epithelial or myoepithelial patterns and transplanted them in syngeneic animals. One stabilized line (EPI) and four cloned lines (A, C, D, E) with epithelial characteristics, confirmed by positive reactions for keratins in immunocytochemical and immunoblot tests, constantly gave rise in vivo to carcinomas, which, however, lacked structural and functional patterns typical of the original tumor. A fusiform shape and immunocytochemical characteristics of myoepithelial cells were observed in three clones (H, I, L), which in vivo gave rise to sarcomatous and mixed carcinosarcomatous neoplasms. These data are consistent with the above hypothesis and indicate that breast carcinomas derive from epithe lial cells, while sarcomatous and carcinosarcomatous neoplasms can originate from myoepithelial cell proliferation. This study provides data suggesting myoepithelial cell involvement in the development of patho logical entities occurring in the human breast and displaying mixed epithelial and stromal neoplastic components, i.e.. cystosarcoma phylloides and sarcomatous metaplasia in carcinomas. Tumor. R3230AC transplantable mammary tumor was obtained from Biomeasure (Hopkinton, MA) and maintained by serial s.c. trans plantations into the dorsal region of female Fischer 344 inbred rats (100-120 g body weight; Nossan, Correzzana, Milan, Italy). Tumor fragments were obtained from nonnecrotic areas of the donor rat, mechanically dispersed in a Potter homogenizer and suspended in RPMI. Approximately 1 mm3 of tumor (or 1-3 x IO6neoplastic cells) INTRODUCTION was used for monthly transplantations. The tumor consistently grew to a 2-em mass within 20 days. Animals were killed by decapitation every 25-35 days; tumor tissue was in part inoculated in recipient rats as described above, in part fixed in absolute ethanol or methacarn for histology and immunohistochemistry (antibodies listed in Table 1), and in part processed for cell culture lines (see below). In order to evaluate the response to estrogen stimulation, tumorbearing animals were given for 1 month weekly s.c. injections of estradici valerate (20 mg/kg body weight) according to the method of Hilf (14). Establishment of Primary Stabilized and Cloned Cultures. The tumor tissue, removed aseptically, was minced into small pieces (~1 mm3) with scalpels and digested with 125 units/mg collagenase (type II) (Worthington Biochemical Corp., Freehold, NJ) in RPMI (GIBCOIsland Biological Co., Grand Island, NY), 10% PCS3 (GIBCO), 200 units/ml penicillin, 200 Mg/ml streptomycin (EUROBIO), and 2.5 mg/ ml Fungizone (GIBCO) for 2 h at 37*C. The R3230AC mammary carcinoma, transplantable in Fischer rats, was originally described by Hilf et al. (1) as a milkproducing adenocarcinoma. Under estrogen stimulation, it dis plays marked increase of milk protein production and cellular vacuolation. The biochemical properties and hormone depend ency of this tumor have been investigated by several authors (2-12). We observed that the peculiar functional secretory similarity of this carcinoma to the normal mammary gland is matched by a parallel similarity in structure; both epithelial and basally located myoepithelial cells are present, outlining neoplastic pseudoglandular structures and showing the typical immuno cytochemical and ultrastructural characteristics of these cells in the normal breast (13). The R3230AC mammary carcinoma therefore represents an experimental model with unique morphological and functional properties. We have established and cloned in vitro cell lines from this tumor and studied their morphology, immunocytochemistry, and function. These cell lines were further investi gated by transplanting them back into syngeneic rats, where they gave rise to carcinomas, sarcomas, and mixed carcinosarcomas. This experimental model provides data that help to understand the significance and genesis of peculiar and still unexplained pathological entities occurring in the human breast, i.e., cystosarcoma phylloides and sarcomatous meta plasia in carcinomas. Received 7/22/91; accepted 1/8/92. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by grants from the CNR (Rome), AIRC (Milan), and the Ministry of the University. 2To whom requests for reprints should be addressed. Single cell suspensions and several small tumor fragments were washed twice in RPMI supplemented with 10% FCS without collagen ase and plated in T25 flasks (Falcon Plastics, Los Angeles, CA) at a concentration of about 3 x 10s cells/flask. Incubation of the flasks for 24 h at 37'C in a 5% CO2 humidified atmosphere allowed most of the fragments and free cells to attach. In order to obtain an epithelial cell line (EPI) from the primary culture, elongated cells were removed following trypsin-EDTA treatment (15) with a minor modification proposed by Kuzumaki et al. (16). Briefly the mixed cell sheet was rinsed once with Ca2+-and Mg2+-free PBS and incubated for 3 min at 37*C in the presence of fresh trypsin-EDTA. The less rapidly detaching cells, mostly islands of epithelium-like cells, were rinsed and supplied with fresh medium. This procedure was repeated every 3 days until epithelial like cells covered more than 90% of the culture surface. The stabilized EPI cells are presently at the 100th passage. Subcul tures were routinely carried out in RPMI, 10% FCS, and antibiotic. Cell Cloning. The mixed population obtained from primary cultures (of R3230AC tumor) as well as the stabilized (EPI) epithelial cells were cloned following the dilution plating technique proposed by Sato et al. (17). A single cell suspension was appropriately diluted and 96-well microtest plates (Falcon) were seeded; each well was inoculated with a cell suspension yielding an average of 1 cell/well. Those wells contain ing only one cell as ascertained by microscope inspection were consid ered available for clone establishment; the others were discarded. Conditioned media were prepared by adding to the routine medium 10% of the supernatant obtained from confluent cultures of epithelial cells filtered through 0.22-/im Millipore filters and stored at 4*C before use. We obtained 3 clones (I, H, L) from the mixed population of primary culture and 4 clones (A, C, D, E) from the EPI cell line. The cell lines, at early passages, were reinjected into syngeneic animals and subcultured until passage 80. 3The abbreviations used are: FCS, fetal calf serum; PBS, phosphate-buffered saline; NTEN, 50 miviTris, pH 7.4-0.15 M NaCI-2 mM EDTA-0.1% Nonidet P40; sm, smooth muscle. 1553 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1992 American Association for Cancer Research. MAMMARY CARCINOMA CELL LINES In Vitro Hormonal Stimulation. Primary cultures, stabilized EPI cells, and clones D and E were cultured in Petri dishes and on glass coverslips for 9 days in a basal medium containing 10 * M 17/3-estradiol (Merck, Darmstadt, Germany) and 10% of charcoal-adsorbed PCS (18). The medium was changed every 3 days. a-Lactalbumin production was estimated immunocytochemically. Light Microscopy. The morphological appearance of living cultures in flasks was observed on a Leitz Labovert inverted microscope fitted with phase contrast. Mixed cells from primary cultures, EPI cells, and clones were also grown on glass coverslips; fixed in methanol; and stained by the Papanicolaou method for cytological examination. For immunohistochemical tests, cells were grown on glass coverslips, fixed in methanol for 5 min at -20'C, permeabilized in acetone for 5 s at —¿20*C, and réhydratée! with pig normal serum (diluted 1:5(1in PBS for 20 min). Cells were incubated at 37'C for 60 min with the primary antibodies listed in Table 1 and then with the appropriate fluoresceinlabeled secondary antisera (Sera-Lab, Ltd., Sussex, England) diluted 1:10 in PBS for 30 min at room temperature, following a standard indirect immunofluorescence procedure. Electron Microscopy. EPI cells and cells from clone I were fixed in a solution of 1-2% glutaraldehyde in 0.2 M cacodylate buffer (pH 7.3) and 2% aqueous osmium tetroxide for l h at 4*C, dehydrated through graded alcohols, cleared in propylene oxide, and embedded in EponAraldite. Ultrathin sections were counterstained with uranyl acetate and lead citrate and examined with a Siemens Elmiskop 1 or a Philips EM 401 electron microscope. Cell Extracts. The established cell line EPI and the clones (C, D, H, I, L) were homogenized in a buffer 20 mM Tris (pH 7.4), 0. l M NaCl, 5 mM MgCl2, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1 mM 2-mercaptoethanol, and 2000 lU/ml Trasylol. Extracts were stored at -80*C until used. Protein concentration was determined by the method of Lowry as modified by Markwell et al. (19). Immunoblotting. Total proteins (100 ng) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis on 8% acrylamide gel slabs and transferred to nitrocellulose filters in a Ployblot apparatus (ABN, Emeryville, CA) according to the manufacturer's instructions. Nitrocellulose was then saturated in 3% bovine serum albumin in NTEN for 3 h at 37'C, incubated in the same buffer containing the anticytokeratin 19 monoclonal antibody 1165 (Amersham Interna tional, England) diluted '/iooor an anti-a-sm-actin monoclonal antibody diluted 1:800 (Sigma Chemical Co., St. Louis, MO) for 16-18 h at 4"C, washed twice for 10 min each in NTEN, incubated with the secondary rabbit anti-mouse antibody (Dako) diluted 1:500 for 3 h at 4"C, washed twice for 10 min each in NTEN, incubated with '"!protein A (0.1 /¿Ci/ml)(Amersham), and then washed as before, air dried, and exposed for 2 days at —¿80°C to Hyperfilm MP autoradiographic films (Amersham). Transplantation of Cell Lines into Animals. Cultured cells (approxi mately 0.8-4 x IO6, suspended in 0.5-0.8 ml RPMI), obtained from early passages (3rd-8th) were injected s.c. into female Fischer rats. The occurrence and time of growth of tumors were recorded. Tumors growing in syngeneic rats from cultured lines were retransplanted into other rats, following the procedure outlined above. Tissue blocks were fixed in methacarn (20) and embedded in paraffin. Sections were routinely stained with hematoxylin and eosin and processed for immunocytochemical characterization using the antibodies listed in Table 1. To detect immunological binding, the avidin-biotinylated peroxidase complex procedure (21) was used, after treatment of the sections with the appropriate biotinylated secondary antisemiti (Vector, Burlingame, CA). In Vivo Hormonal Stimulation. Animals bearing palpable tumors obtained from EPI, D, E, H, and I cells were treated with estrogen following the protocol used in R3230AC-bearing animals (see above). RESULTS Serially Transplanted R3230AC Tumors. The gross and mi croscopic appearances of the R3230AC mammary tumor, de scribed in detail elsewhere (13), were examined at each transplantation. The tumors were mostly well delimited, but focal invasion of the adjacent muscular layers was observed in some cases. Dis tant métastaseswere never recorded in the 52 rats transplanted in this study. Uniform histological patterns were observed with acinar and/or gland-like structures lined by polygonal epithelial cells and basal, elongated myoepithelial cells. The original tumor stained positively for «-sm-aetin in the elongated basal cells, thus confirming their myoepithelial nature (Fig. la). Epithelial secretory cells were stained with different antibod ies to keratin. AE1, a monoclonal recognizing acidic cytokeratins, was positive only in single squamous cells while AE3, a monoclonal to basic cytokeratins, recognized a few epithelial cells and the majority of basal myoepithelial cells. Cytokeratin 8 (M, 52,000-55,000) recognized by 1166 monoclonal was found in 70% of epithelial cells (Fig. \b) while cytokeratin 19 (M, 40,000), marked by 1165 monoclonal, was present in less than 10% of epithelial cells. The basal membrane was positively stained by anti-type IV collagen antibodies. Primary and Stabilized Cell Cultures and Clones. Primary cell cultures, large flat cells with epithelial appearance, were sur rounded by thick bundles of fusiform cells, that tended to overgrow the epithelial-like elements. Cytokeratins were consistently detected in large epitheliallooking cells, while only 10% of elongated cells were positive for the anti-sm-actin monoclonal. The majority (80%) of the cells were vimentin positive. Collagen type IV was present as small dots on the surface of the fusiform cells. Stabilized EPI cells formed closely packed islands of large cuboidal cells, sometimes surrounded by elongated cells (Fig. la). Isolated single cells were fusiform with fibroblast-like appearance (Table 2). All the cells were intensely positive for the 1165 and 1166 monocle muÃ-sagainst cytokeratins. Bundles of intermediate filaments at the periphery of the cytoplasm and in a perinuclear arrangement were stained by the AE3 mono- Table 1 Reagents used in immunohistochemistry and immunofluorescence cellsEpithelialMyoepithelialEpithelialEpithelia AntigenCytokeratin 40,000)Cytokeratin 19 (M, EnglandAmersham International, undiluted1/100-1/101/40-1/11/800-1/1001/350-1/501/300-1/101/400-1/201/5000-1/50" 52,000-55,000)Cytokeratins 8 (M, EnglandCambridge International, 19-16-15-14-10Cytokeratins (MAb)AE3 8-7-6-5-4-3-2-1..-Smooth (MAb)1 actinCollagen muscle IVVimentina-LactalbuminRat type (MAb)AntiserumV9 A4 undilutedI/ '1(1 IO- UnitedStates Research Laboratory, AmericaCambridge of UnitedStates Research Laboratory, AmericaSigmaHeyl, of GermanyDakopatts, Berlin, (MAb)AntiserumAntiserumSourceAmersham DenmarkDr. Glostrup, DOurVonderhaar. Bethesda, M MFGMReagent1165(MAb)1166(MAb)AE1 laboratoryDilution"1. Data referred to dilutions in immunoperoxidase and immunofluorescence, respectively. Ali,1554Target MFGM, milk fat globule membrane; M membraneMesenchymalSecretorySecretorymono antibody. Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1992 American Association for Cancer Research. MAMMARY CARCINOMA CELL LINES were moderately detectable with the 1165 monoclonal antibody directed against keratin 19; actin micro lì laments were clearly detectable in some elements (Fig. 2e). Production of type IV collagen was shown in Clone H, I, and L cells. Specific immunolocalization was locally revealed inside the cytoplasm and patchy' deposits were detectable in the intercellular spaces (Fig. 2f>. The immunocytochemical results were further checked in immunoblots. All clones, as well as the EPI line, when tested by Western blotting for the presence of cytokeratin 19 with the antibody 1165, showed a M, 43,000 band, characteristic of this intermediate filament; only Clone H was always negative for keratin (Fig. 3). Electron Microscopy. The EPI cells showed typical features of epithelial cells with desmosomes and numerous microvilli; bundles of intermediate filaments were present in the cytoplasm of some cells. On the contrary Clone I cells had an elongated shape with large mitochondria and numerous ribosomes; no desmosomes were observed but tight junctions were occasion ally present. Microfilaments were detected, often arranged in bundles close to the cell membrane. Dense bodies along such bundles were observed (Fig. 4). Growth Patterns of Transplanted Clones. EPI cells injected into Fischer rats generated approximately 2 cm tumors within 30 days. Histologically, polygonal or round cells were arranged in gland-like or trabecular structures, delimited by thin stromal bundles (Fig. 5). No actin-rich cells were observed. Necrosis and foci of squamous metaplasia were present. Cytokeratins and rat milk fat globule membrane were abundant in all tumor cells. Type IV collagen and vimentin were not detectable. All seven clones were injected into animals and generated tumors in a time range of 20 days to 7 months (Table 3). Animals were killed when tumors grew up to 2 cm or more (largest diameter) and the tumor material was reinjected into other syngeneic rats for one to three passages. Clone A, D, and E tumors had frank epithelial carcinomatous features with solid nests or gland-like structures haphazardly intermingled with a spindle-shaped cell component. Foci of squamous metaplasia were seen in Clone A and E tumors (Fig. 6). Keratins were found in a variable proportion of carcinoma !f» tous cells, while vimentin stained the stromal component con <• sistently. Few cells reacted with rat milk fat globule membrane antiserum. Type IV collagen was present in limited areas of these tumors; cell membranes of single cells were outlined and some neoplastic nests were marked at the basal membrane level. No actin reactivity was observed. Fig. 1. Original R3230AC rat mammary carcinoma. In a, nests of neoplastic cells are lined by a peripheral layer of flattened actin-rich myoepithelial cells. Tumors obtained from Clones H, I, and L had a sarcomatous Immunoperoxidase staining with a-smooth muscle actin monoclonal, x 250. In appearance with spindle-shaped neoplastic cells mixed with b, immunostaining for cytokeratin 8 (monoclonal 1166) is positive in the majority liposarcoma-like areas (Fig. 7). A second generation tumor, of neoplastic epithelial cells arranged in nests or pseudoglandular structures, x ISO. obtained by previous injection of clone L cells, showed keratinpositive adenocarcinomatous foci while in a tumor originated from Clone H, areas of chondromatous metaplasia were pres clonal in 80% of the cells. The AE1 monoclonal was positive ent. Clone I had a peculiar morphology, strongly resembling in only 40% of the cells. Clones A, C, D, and E (obtained from EPI cells) were cystosarcoma phylloides (Fig. 8, a and b). The epithelial com ponent was strongly positive for cytokeratins and was supported characterized by rounded clusters of cuboidal cells and isolated cells with large cytoplasm (Fig. 2, b and r). Immunohistochemby a highly cellular stromal proliferation reactive to vimentin ically, these clones showed a pattern similar to the EPI cells only. A basal layer of myoepithelial cells lined the ducts, as confirmed by positive actin staining (Fig. 8e). (Table 2). Type IV collagen was positive in myoepithelial cells but not Cells of Clones I, L, and H (all derived from primary cultures in the basal membrane of the phylloides-like tumor from clone of R3230AC tumor) were elongated or star shaped (Fig. 2d). I. Liposarcoma-like tumors were positive for this marker. The cells formed an irregular network on the Petri dish. Gen erally, the cells were proliferating intensely. In Vitro and in Vivo Hormonal Stimulation. Estrogen treat Clone L grew as islands of large star-shaped cells. Keratins ment of control R3230AC tumor induced typical lactational 1555 "•$4 >i. iw?., ms* •¿ %«&3K& &vyK&?r «r.-> ":'-b>;,*,v.*. v^..^:. Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1992 American Association for Cancer Research. MAMMARY CARCINOMA CELL LINES \psr^£â A ; \à » ~ * J^* » . *; •¿,. Fig. 2. /n vitro cultures of the cell lines obtained from R3230AC rat mammary carcinoma. In a, a stabilized cell line (EPI), observed in phase contrast, forms closely packed islands of elongated and cuboidal cells. In b. Clone D cells, observed in phase contrast, grow as small roundish colonies of cuboidal cells. In <•, immunofluorescence for cytokeratins (monoclonal 1165) in Clone D cells gives a strong positive reaction. In d, cells of Clone H, examined in phase contrast, present a star-like shape. In e, intense reaction for «-smoothmuscle actin displayed by cells of clone I.. In/, immunocytochemical detection of type IV collagen in clone H reveals mainly extra- and pericellular deposits, a, e, and d, x ISO; c and e, x 240: / x 480. Table 2 Morphological and immunochemical features 0/R3230AC mammary carcinoma cell lines a-sm-actin++nt IV Vim. 1165-6++ MFGMnt++++ntntnt+ spindle-shapedRoundishRoundishRoundishRoundishStar-likeSpindle-shapedStar-likeRoundish + +++nt -+ nt ++++++ j-+nt—-—++CytokeratinsAE3 —¿â€” ++ ++++++ —¿nt ++ +++nt -+++ nt ++— —¿â€”¿+++ +++ -— -+++ +++ +++ +++++ +++ + spindle-shapedAE1"+nt+ ++Coll. +++ ++Rat cultureMorphologyRoundish °AE1, AE3, 1165, 1166, cytokeratins (see references); Coll. IV, type IV collagen; Vim., \ ¡mentiti;«-sm-actin,«-smooth muscle actin monoclonal; rat MFGM, EPIClone AClone CClone DClone EClone HClone IClone LPrimary serum against rat milk fat globule membrane antigens; EPI, stabilized cell line; nt, not tested. changes and a-lactalbumin production, demonstrated by immunocytochemistry and immunoblotting. In contrast, estrogen stimulation did not affect the morphology of any of the cultured cell lines, and production of a-lactalbumin was never detectable by immunocytochemical procedures. In vivo estrogen stimulation, started as soon as a nodule was palpable, did not induce milk protein production or change the histológica! pattern. DISCUSSION We recently observed that the architectural organization of the R3230AC tumor parallels that of the lactating gland since, in addition to the luminal epithelial (secretory) cells, myoepi- thelial cells are present (13). The neoplastic myoepithelium shows: (a) typical peripheral arrangement against the basement membrane; (b) typical ultrastructural cytoplasmic organization; and (c) the presence of immunocytochemically detectable asmooth muscle actin. This isoform of actin is present, within the epithelial domain, only in myoepithelial cells of the breast and salivary glands (22, 23). Similar biphasic patterns involving both myoepithelial and epithelial neoplastic components have been described in dimethylbenz[a]anthracene-induced mammary tumors in mice (24). Two alternative hypotheses can be advanced to explain the observed phenomena: myoepithelial and epithelial cell com ponents represent differentiated terminal stages stemming from 1556 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1992 American Association for Cancer Research. MAMMARY CARCINOMA CELL LINES o o I - -I 43 Kd - Fig. 3. Sodium dodecyl sulfate-gel electrophoresis of protein extract of the clone cell lines obtained from R3230AC rat mammary adenocarcinoma. Total extracts, run on 12% polyacrylamide gel, were then transferred on nitrocellulose paper and reacted with anti-cytokeratin 1165 monoclonal antibody as described in "Materials and Methods." A strong positive reaction is observed at M, 43,000 (43 Kd) in Clones C and D and EPI, while Clones I and L give a weak positivity and Clone H is negative. a common uncommitted precursor; or they constitute interac tive but independent neoplastic populations. To test these hy potheses, we obtained separate clones of the epithelial and the myoepithelial cells. Primary and stabilized cell populations as well as seven clones were derived from the R3230AC carcinoma; they presented either epithelial or myoepithelial-like features. Transplantation s.c. gave rise to carcinomas, to sarcomas, or to mixed (carcinosarcomatous) neoplasms (Table 3). Epithelial cell lines (EPI, A, D, E) generated in vivo carci nomas lacking both the myoepithelial component and the hor mone responsiveness of the parent tumor; «-lactalbumin pro duction was not stimulated by estrogen administration. A pos sible interpretation of these findings is that the complex structural organization of the primary tumor is necessary to complete functional differentiation. Moreover, a number of investigators have shown that the matrix on which the mam mary epithelial cells attach in vitro modulates their response to hormones (25-29). Myoepithelial-like cells (lines H, I, and L) were elongated; all cells were producing collagen type IV, which is selectively produced in the mammary gland by myoepithelial cells (3032). In addition, tight junctions and subplasmalemmal bundles of microfilaments with interposed dense bodies (characteristic features of myoepithelial cells) (33) were observed in Clone I and a-sm-actin was detected in Clone L. The presence of this isoform of actin in cultured cells is not per se proof of this myoepithelial nature, since it was recently demonstrated in mammary stromal cells evolving into myofibroblasts (34). How ever, in line with the myoepithelial nature of the cloned cells, we detected cytokeratin of basal type by both immunofluorescence and immunoblotting procedures in Clone L and by immunoblotting alone in Clone I cells. All cell lines contained vimentin, but in cultured cells this latter finding is not proof of nonepithelial nature (35) and coexpression of both keratin and vimentin intermediate filaments has been reported in numerous epithelial tumors (36). Tumors generated from cell lines with myoepithelial features showed mainly sarcomatous patterns. Vimentin and collagen type IV were demonstrated in these tumors; the latter finding seems at variance with the behavior of human liposarcomas which do not produce collagen type IV (37). Focal keratin positivity and chondroid metaplasia were found in single tu mors; of major interest was, however, the peculiar association A •¿1 B Fig. 4. Electron microscopic study of Clone I. In 1. the cells show elongated shape and lack desmosomes; the nuclei are irregular in outline. At higher mag nification (B) a selected area (arrows. A) shows subplasmalemmal bundles of microfilaments (arrowheads), with interposed dense bodies. A, x 2,000; B, x 16,000. 1557 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1992 American Association for Cancer Research. MAMMARY CARCINOMA CELL LINES Table 3 Morphological and immunochemical features of in vivo growth ofR3230AC cell lines Cytokeratins .t '.'"'111 VI MU ('l'Ut. Il 1 ITIAEl V^VHli 1165-6EPIClone ' lili. -Carcinomatous —¿â€¢Carcinomatous + ++ ++ ++ +++ ++ nt —¿ -Carcinomatous (adenocarcinoma) —¿Sarcomatous(anaplastic) -metaplasia)Sarcomatous (lipoblasts, with chondromatous + —¿ - ++ ++ - +++ + - + —¿ ++ —¿ +++ ++Sarcomatous(area phylloides-like) (lipoblasts, with Carcinomatous LCarcinomatous —¿areas)+±++ntntnt —¿ —¿ —¿ —¿ ++ ++ AClone CClone DClone EClone HClone IClone Õ T U-3Ill-a\.lIII IVtll ITI! VI AE3 ++ +++ +++ +++ Original R3230AC carcinoma " Coll. IV, type IV collagen; AEl, AE3, 1165, 1166, cytokeratins (see references); Vim., vimentin; a-sm-actin, «smooth muscle actin monoclonal; Rat MFGM, serum against rat milk fat globule membrane antigens; EPI, stabilized cell line; *. not grown after injection; nt, not tested. and provides experimental evidence of derivation of sarcoma tous and mixed carcinosarcomatous tumors of the breast from neoplastic myoepithelial-like cells. The literature provides examples of fibrosarcomas, rhabdomyosarcomas, and other types of sarcoma originating in ani mals transplanted with fragments of spontaneously developed mouse mammary tumors and with long term cultures of mouse mammary cancer cells (16, 40, 41). Peculiarly, mammary tu mors seem to be the only ones for which this phenomenon (of a sarcoma originating by serial transplantation or by culture of an epithelial tumor) has been described. This peculiarity of experimental mammary tumors has some relationship with certain well known features described in human breast neo plasms. Areas of sarcomatous (osteosarcoma, chondrosarcoma) metaplasia are known to occur in carcinomas of the human breast (42); a similar phenomenon is seen rather commonly in malignant mammary tumors of female canines, in which ohmi ciro id elements are regarded as being of myoepithelial derivation (43). Our studies support the hypothesis that these sarcomatous areas might be related to proliferation of neoplastic myoepithe lial cells. Previously, other authors (44, 45) tried to establish cultures of myoepithelial cells from dimethylbenz[a]anthracene-induced rat mammary tumors and from the mammary gland of a neo natal rat. The line called RAMA 25 showed a peculiar sponta neous transformation in vitro from cuboidal to elongated fusi form cells which have been regarded as the equivalent of the >• ..•.. .. A---'- v * „¿-• % —¿. vi,-:' ^ Fig. 5. Tumor derived from EPI cells. Pseudoglandular structures and laminae of polygonal epithelial cells are arranged in a cellular stroma. H&E, x 150. . JK * -*»-.* ..- !'^v * t Fig. 6. Carcinoma derived from Clone E. Solid nests and pseudoglandular structures are separated by thin stromal bundles. Squamous metaplasia can be focally observed, x 150. in Clone I tumors of liposarcomatous areas with features of cystosarcoma phylloides, a lesion with mixed epithelial and stromal growth regarded as the malignant counterpart of the more common fibroadenoma of the breast. Liposarcomatous differentiation is rather common in human malignant phyl loides tumors (38). Ultrastructural and immunocytochemical evidence of myoepithelial derivation of the stromal sarcomatous component in cases of human cystosarcoma phylloides has been reported in the literature (39). Our study confirms these data Fig. 7. Tumor with liposarcomatous patterns derived from Clone L infÃ-ltrales muscle layer of the dorsal region of a Fischer rat. Spindle-shaped cells with hyperchromatic nuclei are intermingled with polygonal vacuolated cells resem bling lipoblasts. x 150. 1558 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1992 American Association for Cancer Research. .v."''Ã-. ; !;î •¿â€¢â€¢ •¿â€¢ m** MAMMARY CARCINOMA CELL LINES Fig. 8. Tumor derived from Clone I. At low magnification (a), the typical architecture of cytosarcoma phylloides is evident. Leaf-like projections of sarcomatous stromu are lined by epithelium. At high power (b) gland-like structures are lined by a double layer of epithelial and myoepithelial cells. Spindle-shaped cells with hyperchromatic and irregular nuclei can be observed in the strenna. In c, immunostaining for a-smoolh muscle actin reveals a regular layer of actin-rich myoepithelial cells basally located in gland-like structures. No staining is detectable in the spindle-shaped sarcomatous cells, a, x 70; b and c,x 150. normal myoepithelial cells (32, 46, 47). Formation of fusiform cells has also been described in cell lines derived from BALB/ CDH mouse mammary tumors (48) and from nitrosomethylurea-induced mammary tumors in WF rats (49). According to the observations of Dulbecco et al. (49) and Warburton et al. (32) these fÃ-broblast-like cells did not exactly correspond to myoepithelial cells but showed immunocytochemical features common to both stromal cells (presence of vimentin and of Thy-1-1 antigen) and myoepithelial cells (production of type IV collagen). The biphasic behavior of myoepithelial cells in skin and salivary gland tumors, where these cells seem to be able to form a stromal matrix, has been described and discussed by several authors (50, 51). The nature of the myoepithelial cell itself is indefinite: while its content of basic-type keratins sug gests an epithelial nature, the presence of vimentin (52) and its contractile ability, testified to by the presence of bundles of «smooth muscle actin (a unique feature among epithelial cells), indicate its affinity to mesenchyme-derived cells. Even the assumption that myoepithelial cells are terminally differen tiated cells (53, 54) seems untenable, since we observed (55) that in the developing mouse mammary gland myoepithelial cells can proliferate. All these data, in line with the reported experimental obser vation in R3230AC rat tumor and related cell lines, support the hypothesis that mixed epithelial-stromal tumors of the mammary gland can originate from the myoepithelial cells. ACKNOWLEDGMENTS We thank Professor P. M. Cullino (University of Turin, Turin, Italy) and Professor L. Ozzello (Columbia University, New York, NY) for criticisms. REFERENCES 1. 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Sapino, A., Macri, L., Gugliotta, P., and Bussolati, G. Immunohistochemical identification of proliferating cell types in mouse mammary gland. J. Histo chem. Cytochem., 38: 1541-1547, 1990. 1560 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1992 American Association for Cancer Research. Tumor Types Derived from Epithelial and Myoepithelial Cell Lines of R3230AC Rat Mammary Carcinoma Anna Sapino, Mauro Papotti, Brunella Sanfilippo, et al. Cancer Res 1992;52:1553-1560. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/52/6/1553 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. 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